ABSTRACT This application is being submitted in response to NOT-AI-18-055. Under a NIDA Avant Garde Award, we have developed a candidate heroin vaccine which induces antibodies that bind heroin/opioids and, subsequently, prevent the drug from crossing the blood-brain barrier and interacting with the brain's -opioid receptor. Pre-clinical testing of the vaccine candidate in mice and rats demonstrated protection from subcutaneous (SC) and intravenous (IV) heroin challenge. The binding affinities of the antibodies to heroin and other abused prescription opioids were very tight (<0.1-15 nM). Recently, we were awarded a UG3 grant (1UG3DA048351-01) to advance our vaccine candidate toward a Phase 1/2a human clinical trial. The clinical trial design proposed testing the vaccine in two cohorts. The first was normal healthy volunteers, who would be challenged with morphine following completion of immunization. The second cohort was heroin users, who have been abstinent for at least 6 months. While the grant proposal was under review the FDA issued draft guidance that stated substance abuse therapeutics should be tested in population for which the therapeutic is intended and not in normal healthy volunteers. One of the goals of this supplemental application is hold an INTERACT (pre-pre-IND) meeting with the FDA to discuss the cohort design of the Phase 1 clinical trial. We propose to have this meeting within year 1 of the parent proposal to better guide us in the preparation of the pre-IND in year 2 of the proposal. Another goal of this supplemental application is to compare the adjuvants ALFQ (Army Liposome Formulation containing monophosphoryl lipid A (MPLA) and QS-21) and ALFQA (ALFQ with aluminum hydroxide) with the adjuvant ALFA (Army Liposome Formulation with MPLA and aluminum hydroxide) that has been selected for the Phase 1 clinical trial. ALFQ was developed as potent adjuvant that is similar to GSK's AS01B adjuvant that is used in the highly successful SHINGRIX vaccine for shingles. However, AS01B has a high rate of side effects including sore muscles, mild fever, and mild influenza-like symptoms, while studies in animals including non-human primates and rabbit pharmacology-toxicology studies have not revealed these adverse side effects. In this supplement, we will conduct mouse studies with the TT-6-AmHap heroin vaccine that is mixed with ALFA, ALFQ and ALFQ. The vaccine formulations will be evaluated for efficacy against repeat-dose heroin challenges. Antibodies will be analyzed for titer, cross-reactivity and IgG subclass by ELISA. Antibody affinities will be measured by microscale thermophoresis. Spleen B cells, bone marrow plasma cells and circulating plasma cells will be obtained following both primary and boosting immunizations and analyzed by ELISPOT and flow cytometry. The overall outcome of this portion of the supplement will be to identify an adjuvant that induces antibodies of the appropriate IgG subclass distribution that supports the highest vaccine efficacy. In addition, the role of the adjuvants, ALFA, ALFQ and ALFQA, in the level of induction and sustenance of B memory cells and plasma cells, and their relationship to vaccine efficacy against heroin challenge will be established.